HIV-1 replication is a combined result of host-virus interactions occurring at multiple steps of the virus life cycle including entry, gene expression, assembly, and budding. It is our long-term goal to understand regulation of HIV-1 gene expression and replication by host factors, thereby develop strategies for treating AIDS patients and ultimately preventing and eliminating HIV-1 infection. Unlike primary CD4+ T lymphocytes and macrophages/momocytes, CD4- astrocytes are less susceptible to HIV-1 infection and do not support post- entry productive HIV-1 replication. Therefore, astrocytes have been regarded as an ideal natural platform to define the host factors that are essential for HIV-1 entry, gene expression and replication. Using a retrovirus- based expression cDNA library, we have revealed that HIV-1 gains access to astrocytes through human mannose receptor in a relatively inefficient CD4-independent and endocytosis-dependent manner. In addition, using the subtractive cloning strategy, we have identified Sam68 to be, at least in part, responsible for post- entry non-productive HIV-1 replication in astrocytes. The overall goal of this proposal is to characterize the molecular mechanisms of Sam68 function in HIV-1 replication in astrocytes as well as other HIV-1 natural target cells. This proposal is based on our three major findings (1) Sam68 is an essential cellular co-factor for HIV-1 Rev nuclear export; (2) Inhibition of HIV-1 replication by Sam68 mutants lacking a nuclear localization signal correlates with the cytoplasmic localization of constitutive Sam68; (3) Sam68 regulates translation of HIV-1 mRNAs in cytoplasm. The underlying hypothesis for this proposal is that Sam68 regulates HIV-1 replication at multiple steps of viral life cycle in both the nucleus and the cytoplasm. In other words, Sam68 is an important cellular co-factor for HIV-1 replication and pathogenesis. To test this hypothesis, we propose three interrelated specific aims: (1) To determine the relationship between Sam68 expression and HIV-1 replication; (2) To characterize Sam68 function in HIV-1 Rev nuclear export; and (3) To define the cytoplasmic function of Sam68 in HIV-1 replication. We will use a variety of state-of-art biochemical, cellular, and molecular approaches throughout the studies. The answers sought have fundamental significance for understanding of this critical and pervasive protein Sam68 in HIV-1 replication. They should also aid in the development of anti- HIV therapeutic strategies. PUBLIC HEALTH RELEVANCE: HIV-1 infection often causes a number of brain diseases and affects the ability of people to care for themselves and thus the quality of their daily life. The social and economic impact can not be overemphasized. The current study seeks to have a better understanding of the host factors that are required for HIV-1 propagation and then use the knowledge acquired to develop new treatment options for HIV/AIDS patients.